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Originally published In Press as doi:10.1074/jbc.M111290200 on February 4, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13778-13786, April 19, 2002
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Exposure of Cryptic Domains in the alpha 1-chain of Laminin-1 by Elastase Stimulates Macrophages Urokinase and Matrix Metalloproteinase-9 Expression*

K. M. Faisal KhanDagger , Gordon W. Laurie§, Timothy A. McCaffrey, and Domenick J. FalconeDagger ||**

From the Dagger  Department of Pathology, || Department of Cell Biology and Anatomy, and the Vascular Biology Center, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, the § Department of Cell Biology, University of Virginia, Charlottesville, Virginia, and the  George Washington University Medical Center, Washington, D. C.

Received for publication, November 27, 2001, and in revised form, January 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha 1:Ser2091-Arg2108, a peptide derived from the alpha 1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPKerk1/2, leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha 1:Ser2091-Arg2108 is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha 1-chain including alpha 1:Ser2091-Arg2108 and the globular domain. A peptide from the first loop of the globular domain (alpha 1:Ser2179-Ser2198) triggered the phosphorylation of MAPKerk1/2 and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha 1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha 1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The synthesis and activation of serine and matrix metalloproteinases (MMP)1 by monocytes and macrophages play an important role in their migration through extracellular matrix (ECM) and clearance of extravascular fibrin and necrotic debris (1-7). In earlier studies, we tested the hypothesis that the ECM regulates macrophage proteinase expression by culturing macrophages on ECM purified from Engelbreth Holm Swarm (EHS) sarcoma (MatrigelTM) (8, 9). Results demonstrated that the expression of urokinase type plasminogen activator (uPA) and MMP-9 by murine RAW264.7 macrophages, human THP-1 monocytes, and human bone marrow-derived macrophages were strongly up-regulated. This was the first demonstration that the engagement of ECM by macrophages stimulates their expression of both uPA and MMP-9. Since the uPA/plasmin system is a physiologic activator of MMPs (10, 11), the ECM emerges as a potent regulator of the macrophage-degradative phenotype.

The ECM component responsible for stimulating macrophage proteinase expression was identified as laminin-1 (8). Laminins are large heterotrimeric molecules (~500-1000 kDa) with multiple domains that mediate their attachment to cells and other ECM components (12). Twelve laminin heterotrimers (assembled from five alpha , three beta , and three gamma  chains) have been identified in mammals. Laminin-1 was the first of this family to be identified (13) and remains the best understood of the laminin isoforms (12, 14). It consists of alpha 1 (~400 kDa), beta 1 (~200 kDa), and gamma 1 (~200 kDa) chains. The NH2-terminal portions of the alpha 1-, beta 1-, and gamma 1-chains are free, whereas much of the rest of the chains are twisted in a coiled-coil. The COOH-terminal portion of the alpha 1-chain extends past the coiled-coil region and forms a large oblong globule (G-domain) consisting of five homologous repeats. The G-domain is the principle heparin-binding region of laminin-1 (15, 16).

In an effort to identify the domains of laminin-1 responsible for stimulating macrophage proteinase expression, we examined synthetic peptides, which were reported to support cell adhesion and stimulate a variety of biological responses. Incubation of RAW264.7 macrophages and THP-1 monocytes with alpha 1:2099SIKVAV2104 stimulated their expression of both uPA and MMP-9 (8). Neither a scrambled alpha 1-chain peptide nor beta 1-chain peptides had any effect on macrophage proteinase expression. Thus, a peptide derived from the alpha 1-chain of laminin-1 stimulates both uPA and MMP-9 expression by macrophages.

ECM components contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules. For example, it was recently reported that a cryptic domain in laminin-5 that stimulates cell motility is exposed following cleavage with MMP-2 or MT1-MMP (17, 18). The synthetic laminin-1 peptide alpha 1:2099SIKVAV2004 that stimulates macrophages uPA and MMP-9 (8) is derived from the region of the alpha 1-chain associated with the coiled-coil and is probably not exposed in the intact molecule (16). Therefore, we compared the ability of intact laminin-1 and alpha 1-chain peptide to regulate macrophage proteinase expression. Results of those experiments demonstrated that intact laminin-1 had no effect on macrophages' proteinase expression, whereas uPA and MMP-9 expression were stimulated in a dose-dependent manner by alpha 1:2091SRARKQAASIKVAVSADR2108 (9). Moreover, incubation of macrophages with the alpha 1-chain peptide, but not intact laminin-1, triggers a phosphorylation cascade resulting in the activation of protein kinase C, which in turn leads to the activation of MAPKerk1/2. The observed signaling events were causal to the induction of proteinase expression, since inhibition of tyrosine kinases, protein kinase C, or MEK-1 (MAPK kinase) blocked the ability of alpha 1:2091SRARKQAASIKVAVSADR2108 to induce uPA or MMP-9 expression (9). Taken together, these data suggest that a cryptic domain of laminin-1 induces a signaling pathway distinct from intact laminin-1 and up-regulates macrophage proteinase expression. The unmasking of this cryptic domain may play a role in regulation of macrophage degradative phenotype and tissue remodeling.

In studies reported here, we have sought to identify the proteinases responsible for exposing the domain(s) in laminin-1 that regulate the macrophage-degradative phenotype. Results demonstrate that laminin-1 is susceptible to cleavage by a variety of proteinases including elastase, MMP-2, MMP-3, MMP-7, and plasmin. However, only cleavage by elastase generated fragments that stimulated proteinase expression by RAW264.7 macrophages and thioglycollate-elicited macrophages. Laminin fragments were fractionated by affinity chromatography on heparin-Sepharose. Heparin binding fragments stimulated macrophage proteinase expression severalfold greater than fragments that did not bind, suggesting that the stimulatory domains were in or associated with the G-domain. A peptide from the first globular repeat in the G-domain (alpha 1:Ser2179-Ser2198; SN peptide), which plays a role in the adhesion of cells to laminin-1 (19), triggered the phosphorylation of MAPKerk1/2 and stimulated the expression of macrophage uPA and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha 1-chain and stimulated macrophage proteinase expression. When immunodepleted with anti-alpha 1-chain IgG, the heparin-binding fraction no longer stimulated proteinase expression. Taken together, we conclude that the COOH-terminal portion of the alpha 1-chain of laminin contains cryptic domains that are exposed by selective proteolysis and stimulate macrophages' proteinase expression.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Murine RAW264.7 macrophages (20) were obtained from American Type Culture Collection. Cells were maintained as adherent cultures in Roswell Park Memorial Medium (RPMI; without HEPES) supplemented with 10% Cellect Gold fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), and 4 mM glutamine (Invitrogen). Experiments to determine the effect of laminin fragments and peptides on macrophage proteinase expression were carried out in macrophage serum-free medium (MSFM; Invitrogen).

Isolation of Peritoneal Macrophages-- Thioglycollate-elicited peritoneal macrophages were obtained from Swiss Webster mice by the method of Edelson and Cohn (21) as described previously (2). Mice were injected intraperitoneally (3 ml/mouse) with 3% brewer thioglycollate medium containing 0.3 mM thioglycollate (Difco). 4 days later, cells were harvested by lavage with cold DPBS. Peritoneal cells were recovered by centrifugation and resuspended in RPMI-10% FBS and plated into appropriate wells. Cells were allowed to adhere for 2 h and then washed free of nonadherent cells.

Proteolysis of Laminin-1-- Murine laminin-1 (BD Biosciences) was incubated with bovine pancreatic elastase (Sigma), active recombinant human MMP-2 (Calbiochem), active recombinant human MMP-7 (Calbiochem), or human plasmin (American Diagnostica). Incubation conditions were as follows. 27 nM laminin-1 was incubated with 0.48 nM elastase for 2.5-120 min in 0.05 M ammonium bicarbonate buffer, pH 7.9; 5.8 nM laminin-1 was incubated with 50 nM MMP-2 for 2-24 h in DPBS; 5.8 nM laminin-1 was incubated with 50 nM MMP-7 for 2-24 h in DPBS; and 22 nM laminin-1 was incubated with 235 nM plasmin in DPBS for 2-24 h. Following incubation, 6× SDS-sample buffer containing 5% beta -mercaptoethanol was added to the samples and boiled for 3 min.

Western Blot for Laminin-1-- In experiments to monitor the degradation of murine laminin-1 by selected proteinases, intact laminin-1 and degraded laminin-1 were electrophoresed in 4-15% polyacrylamide gradient gels under reducing conditions. Proteins were transferred to a PVDF membrane, following which the membrane was blocked in TTBS containing 5% dry defatted milk for 1 h. Following one wash (15 min) in TTBS, the membrane was incubated 1 h with 1.0 µg/ml rabbit anti-murine laminin-1 (Collaborative Biomedical Products) in TTBS containing 3% dry defatted milk. The membrane was washed (twice in TTBS) and reblocked in TTBS containing 5% dry defatted milk for 15 min. The membrane was then incubated 1 h with biotinylated rabbit anti-mouse IgG (1:10,000; Pierce), washed (twice in TTBS) and incubated for 1 h with preformed avidin-biotin-horseradish peroxidase complexes (Pierce) in DPBS plus 0.1% Tween 20. Bound HRP was visualized utilizing enhanced chemiluminescence.

Heparin-Sepharose Chromatography of Elastase-derived Laminin Fragments-- Laminin-1 (5 mg) was digested with elastase (2.5 µg) at 4 °C for 1 h and room temperature for 20 h in 50 mM ammonium bicarbonate buffer, pH 7.9. Proteolysis was stopped by the addition of excess phenylmethylsulfonyl fluoride. The sample was loaded on a heparin-Sepharose column (5 ml), which was previously equilibrated with ammonium bicarbonate buffer. Fractions of 1 ml were collected and monitored for the presence of protein by UV spectrophotometry. Unbound laminin fragments were washed from the column with buffer. Bound fragments were eluted with 0.5 M NaCl in ammonium bicarbonate buffer. Peak fractions were concentrated by ultrafiltration and dialyzed against DPBS at 4 °C.

Domain Mapping of Laminin Fragments-- Following elastase digestion and fractionation by heparin-Sepharose chromatography, laminin fragments were electrophoresed in 4-15% polyacrylamide gradient gels under reducing conditions. Proteins were transferred to a PVDF membrane, following which the membrane was blocked in TTBS containing 5% dry defatted milk for 1 h. Following one wash (15 min) in TTBS, the membrane was incubated with either a rabbit antibody directed against a recombinant protein corresponding to amino acids 1856-2099 of the alpha 1-chain of laminin-1 (Santa Cruz Biotechnology) or a rabbit antibody directed against the COOH-terminal 50-kDa portion of the G-domain (16) (RG50; kindly provided by Peter Yurchenco, UMDNJ) in TTBS containing 3% dry defatted milk. The membrane was washed (twice in TTBS) and reblocked in TTBS containing 5% dry defatted milk for 15 min. The membrane was then incubated 1 h with biotinylated goat anti-rabbit IgG (1:10,000; Pierce), washed (twice in TTBS), and incubated for 1 h with preformed avidin-biotin-HRP complexes (Pierce) in DPBS plus 0.1% Tween 20. Bound HRP was visualized utilizing enhanced chemiluminescence.

Preparation of Cell Lysates-- RAW264.7 macrophages were lysed in Tris buffer, pH 7.5, containing 20 mM Tris-HCl, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM beta -glycerophosphate, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotonin. Lysates were centrifuged (14,000 × g) for 20 min at 4 °C. The supernatants were recovered, normalized for protein, and mixed with SDS sample buffer with beta -mercaptoethanol and boiled for 5 min. Equal amounts of cell lysates were applied to gels based on protein content.

Western Blot Identification of Phosphorylated MAPKerk1/2-- Cell lysates were electrophoresed in 4-15% polyacrylamide gradient gels. Proteins were transferred to a PVDF membrane, following which the membrane was placed in blocking buffer for 1 h. Following one wash in PBS, the membrane was incubated 1 h in blocking buffer containing 0.1 µg/ml rabbit anti-phosphospecific p44/p42 MAP kinase IgG (New England Biolabs). The membrane was washed (twice in TPBS) and incubated for 1 h in blocking buffer containing 0.3 µg/ml goat anti-rabbit IgG conjugated to HRP (Transduction Laboratories). The membrane was washed in TPBS (three times) followed by PBS (once). Bound HRP was visualized utilizing enhanced chemiluminescence.

Determination of Plasminogen Activator Activity-- Plasminogen activator activity was quantitated utilizing a sensitive functional assay for plasmin (22). Aliquots of conditioned media were added to microtiter wells containing 82 µl of DPBS plus 0.05% Tween 20, 13 µg of the plasmin substrate D-Val-Leu-Lys-amino methyl coumarin (Enzyme Systems Products), and 0.5 µg of bovine plasminogen (American Diagnostica). Samples were mixed and incubated at 37 °C for 2.5 h. Cleavage of the substrate was monitored by measuring the increase in fluorescence in a Fluoroscan microplate reader (excitation: 330-380 nm; emission: 430-530 nm). Concentrations of uPA in the test samples were extrapolated from a standard curve utilizing high molecular weight uPA (American Diagnostica). Plasminogen activator activity in macrophage-conditioned media was completely inhibited when preincubated with a polyclonal anti-human uPA IgG (American Diagnostica).

Determination of Metalloproteinase Activity-- The presence of metalloproteinase activity in cellular conditioned media was determined utilizing enzyme zymography as previously described (8). Conditioned media were mixed with SDS sample buffer (without mercaptoethanol) and incubated for 30 min at 37 °C. Samples and molecular weight markers were electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. The gel was then washed (twice) in 2.5% Triton X-100 to remove SDS. The gel was incubated at 37 °C for 48 h in 200 mM NaCl containing 40 mM Tris-HCl and 10 mM CaCl2, pH 7.5, and stained with Coomassie Blue. The presence of gelatinolytic activity was identified as clear bands on a uniform blue background following destaining.

Northern Blot for MMP-9 mRNA Levels-- Total RNA was isolated from RAW264.7 macrophage as previously described (23). The poly(A) mRNA fraction was isolated utilizing the Poly(A)Ttract® mRNA isolation system (Promega, Madison, WI) according to the manufacturer's instructions. Samples were electrophoresed in agarose, transferred to nylon membrane (Schleicher and Schuell), and hybridized with a 32P-labeled murine cDNA for MMP-9 (24) (kindly provided by Dr. G. Opdenakker, Rega Institute for Medical Research, University of Leuven, Belgium).

Isolation and Identification of Laminin Fragments from Human Aortic Aneurysm-- A specimen of surgically removed abdominal aneurysm (~4 g, wet weight) was dissected free of fat and thrombus, minced, and extracted with 4 ml of 5 M urea in 50 mM Tris, pH 7.12, containing 10 mM EDTA for 6 h at room temperature and overnight at 4 °C. Tissue pieces were removed from the extract by centrifugation and clarified by filtration (0.45 µm). The extract was applied to a heparin-Sepharose column equilibrated with the extraction buffer without EDTA. Unbound proteins (peak 1) were washed from the column with extraction buffer, and bound proteins (peak 2) were eluted with 0.5 M NaCl in extraction buffer. Peaks 1 and 2 were dialyzed against DPBS and stored at -20 °C. The unfractionated tissue extract, peak 1, and peak 2 were electrophoresed in 4-15% polyacrylamide gradient gels under reducing conditions. Proteins were transferred to a PVDF membrane and probed with rabbit antibody directed against a recombinant protein corresponding to amino acids 1856-2099 of the alpha 1-chain of laminin-1 (Santa Cruz Biotechnology) as described above.

Immunodepletion of alpha 1-Chain Fragments from Aortic Aneurysm Extract-- Peak 1 and 2 proteins were incubated with rabbit polyclonal directed against alpha 1:1856-2099 of laminin-1 at room temperature for ~4 h with mixing. Protein A-Sepharose (Amersham Biosciences; 100 µl of 10% suspension) was added to extracts, which contained rabbit polyclonal antibodies, and was incubated for 1 h with end over end mixing. Protein A-Sepharose was removed from the suspension by brief centrifugation. The supernatants were collected and diluted 1:3 with RPMI medium.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Elastase-generated Fragments of Laminin-1 Stimulate Macrophage Proteinase Expression-- Laminin family members are susceptible to cleavage by a variety of proteinases including elastase (15, 25), MMP-2 (17, 26), MMP-7 (27), and plasmin (28, 29). To directly test whether cleavage of laminin-1 exposes cryptic domains, which subsequently stimulate macrophage proteinase expression, we incubated intact murine laminin-1 with elastase. Degradation of laminin-1 was monitored by SDS-PAGE followed by Coomassie Blue staining and/or Western blot with polyclonal anti-murine laminin-1. Under reducing conditions, intact laminin-1 (Fig. 1A) appears as two bands: the alpha 1-chain (~400 kDa), and beta 1/gamma 1-chains, which co-migrate (~200 kDa). As reported by others (15, 25), several fragments of laminin-1, ranging from 200 to 20 kDa, are generated by elastase cleavage. The degradation of laminin-1 by elastase was further monitored utilizing Western blot (Fig. 1B). Following a 10-min incubation, immunoreactive alpha 1-chain disappeared, and a lower molecular weight band appeared below the beta 1/gamma 1 chains. At 20 min, immunoreactive beta 1/gamma 1 chains were reduced in intensity and disappeared by 30 min. Following 120 min, immunoreactivity with the polyclonal anti-laminin-1 antibody was lost despite the presence of a range of laminin fragments (Fig. 1A).


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  		Fig. 1.   Elastase-generated fragments of laminin-1. Laminin-1 (27 nM) was incubated with elastase (0.48 nM) at 37 °C for 2.5-120 min in 0.05 M ammonium bicarbonate buffer, pH 7.9. Degradation of laminin-1 was monitored by SDS-PAGE under reducing conditions (A) and Western blot utilizing polyclonal anti-laminin-1 (B).

We next determined whether elastase-generated fragments of laminin-1 would stimulate macrophage proteinase expression. uPA and MMP levels in macrophage conditioned media were quantitated utilizing a fluorescent bioassay and zymography, respectively. As seen in Fig. 2, an overnight incubation of RAW264.7 macrophages with elastase-generated fragments of laminin-1 stimulated their expression of both uPA and MMP-9 to levels achieved utilizing the previously reported stimulatory peptide alpha 1:Ser2091-Arg2108. In contrast to either elastase-degraded laminin-1 or the alpha 1-chain peptide, intact laminin-1 has no effect on macrophages' proteinase expression. These data are proof of the principle that proteolysis of laminin-1 generates fragments that regulate macrophage proteinase expression.


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  		Fig. 2.   Elastase-generated laminin-1 fragments up-regulate macrophage uPA and MMP-9 expression. RAW264.7 cells were suspended in RPMI containing 10% FBS and aliquoted into 96-well plates (105/well). Following 4-6-h adherence, cells were washed to remove serum, and media were replaced with MSFM (Invitrogen) alone or MSFM containing intact laminin (50 µg/ml), alpha 1:2099SIKVAV2104 (100 µg/ml), or elastase-generated laminin fragments (50 µg/ml). The next day, media were recovered and assayed for uPA and MMP-9 activities as described under "Materials and Methods." The uPA data represent the means ± S.E. of three individual wells.

We next determined whether the exposure of stimulatory domains in laminin-1 by elastase was specific. For this purpose, the effect of fragments of laminin-1 generated by MMP-2, MMP-7, or plasmin on macrophage proteinase expression was determined. As seen in Fig. 3, the alpha 1-chain of laminin-1 was degraded by active recombinant MMP-2, MMP-7, and plasmin. However, fragments generated by these proteinases failed to stimulate macrophages uPA or MMP-9 expression (data not shown).


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  		Fig. 3.   Degradation of laminin-1 by MMP-2, MMP-7, and plasmin. Laminin-1 was incubated with active recombinant human MMP-2, active recombinant human MMP-7, or human plasmin as described under "Materials and Methods." Degradation was monitored by Western blot utilizing polyclonal anti-laminin-1.

These data demonstrate that selective digestion of laminin-1 produces fragments that stimulate macrophage proteinase expression. However, in the inflammatory setting, laminin-1 would be subjected to a variety of proteinase, which may modify the elastase-generated fragments to produce biologically inactive fragments. To determine whether the stimulatory activity of elastase-generated laminin-1 fragments is sensitive to other proteinases, we incubated elastase-generated fragments with either 50 nM recombinant MMP-2 or MMP-7 for 4 h and tested their ability to up-regulate uPA expression. Conditioned media recovered from control RAW264.7 macrophages contained 77 ± 3 milliunits of uPA/105 cells (n = 3; means ± S.E.). uPA expression by macrophages incubated (18 h) with elastase-generated fragments increased to 285 ± 25 milliunits/105 cells. Elastase-generated fragments incubated with either MMP-2 or MMP-7 stimulated macrophage uPA expression to 221 ± 21 and 263 ± 24 milliunits/105 cells, respectively. Thus, incubation of elastase-generated fragments with either MMP-2 or MMP-7 had little effect on their ability to stimulate macrophage uPA expression.

Heparin-binding Fragments of Laminin-1 Exhibit Enhanced Proteinase-inducing Activity-- The principal heparin-binding region of laminin-1 has been mapped to the COOH terminus of the alpha 1-chain that forms five homologous loops (G-domain) (30, 31). The synthetic alpha 1-chain peptide Ser2091-Arg2108, which was previously reported to stimulate macrophage uPA and MMP-9 expression (9), is derived from a region of the alpha 1-chain immediately proximal to the G-domain. Therefore, we determined whether affinity to heparin could be utilized as a method to isolate laminin-1 fragments that stimulate macrophage proteinase expression. For this purpose, laminin-1 was digested with elastase, and applied to a heparin-Sepharose column (Fig. 4, top). Unbound laminin fragments were eluted with wash buffer (peak 1), and bound fragments were eluted with 0.5 M NaCl in wash buffer (peak 2). When examined by SDS-PAGE under reducing conditions (Fig. 4, bottom), peak 1 contained a single prominent band (250 kDa), and peak 2 contained three bands (125, 50, and 30 kDa). The laminin fragments in peak 1 and 2 were examined for reactivity with a polyclonal antibody directed against a sequence in the tail of the alpha 1-chain (amino acids 1856-2099) that overlapped the peptide sequence previously reported to stimulate proteinase expression (Fig. 4, bottom). Predictably, the intact alpha 1-chain of undigested laminin was strongly immunoreactive reactive with the anti-alpha 1:1856-2099 antibody. The anti-peptide antibody failed to react with the laminin fragment(s) that did not bind heparin (i.e. peak 1). In contrast, a single immunoreactive band was observed at 120 kDa in fragments that bound to and were eluted from the heparin column (peak 2). Similarly, proteins in peaks 1 and 2 were examined for reactivity with anti-RG50, a polyclonal antibody directed against the terminal 50-kDa portion of the globular domain of laminin-1. As expected, the intact alpha 1-chain of undigested laminin was strongly immunoreactive with anti-RG50. Anti-RG50 failed to react with peak 1 laminin fragments but did react with a heparin binding fragment of ~50 kDa in peak 2. Based on these data, we conclude that peak 2 proteins are derived from the tail region of laminin. Moreover, one of these fragments (120 kDa) contains the sequence previously reported to stimulate macrophages' proteinase expression.


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  		Fig. 4.   Isolation and characterization of heparin-binding laminin-1 fragments. Elastase-digested laminin-1 was fractionated on a heparin-Sepharose column as described under "Materials and Methods." Unbound laminin fragments were washed from the column with wash buffer (peak 1). Bound fragments were eluted with wash buffer containing 0.5 M NaCl (peak 2). Fractions 4-6 of peak 1 and fractions 22-24 of peak 2 were pooled and concentrated. Intact laminin-1, peak 1, and peak 2 were analyzed by SDS-PAGE (Coomassie Blue (CB)) and Western blot utilizing polyclonal antibodies directed against alpha 1-chain amino acids 1856-2099 (anti-alpha 1:1856-2099) and the COOH-terminal 50-kDa portion of the G-domain (anti-RG50).

To determine whether unbound and bound laminin fractions differentially affected uPA expression, peaks 1 and 2 were dialyzed with DPBS and incubated with RAW264.7 macrophages (50 µg/ml) overnight. As seen in Fig. 5, incubation of macrophages with alpha 1:Ser2091-Arg2108 stimulated their expression of uPA, whereas intact laminin-1 had no effect. Following digestion with elastase, both unbound and bound laminin fractions stimulated macrophage uPA expression relative to control cells. The heparin-binding laminin fragments (peak 2) stimulated uPA expression 3-fold more than fragments that did not bind heparin.


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  		Fig. 5.   Enhanced stimulation of macrophage uPA expression by heparin-binding laminin-1 fragments. RAW264.7 cells were suspended in RPMI containing 10% FBS and aliquoted into 96-well plates (105/well). Following 4-6-h adherence, cells were washed to remove serum, and media were replaced with MSFM alone or MSFM containing intact laminin (50 µg/ml), alpha 1:2099SIKVAV2004 (100 µg/ml), peak 1 laminin fragments, or peak 2 laminin fragments (50 µg/ml). The next day, media were recovered and assayed for uPA activities as described under "Materials and Methods." The uPA data represent the means ± S.E. of three individual wells.

A Peptide from the G-domain of Laminin-1 (SN Peptide; alpha 1:Ser2179-Ser2198) Stimulates Macrophages' Proteinase Expression-- In addition to mediating the binding of laminin-1 to heparin, the G-domain contains the epitopes that support cell adhesion. In this regard, alpha 1:Ser2179-Ser2198 (SN peptide), which is located in the first loop of the G-domain, is responsible for the binding of a variety of cells to laminin-1 (19, 32, 33). Another G-domain peptide (alpha 1:Asn2183-Gly2194; AG-10), which comprises the central portion of the SN peptide, stimulated the invasion of cross-linked gelatin films by melanoma cells (34). Therefore, we determined the effect of alpha 1:Ser2179-Ser2198 on macrophages' proteinase expression. As seen in Fig. 6, macrophage uPA expression was increased ~8-fold following overnight incubation with the SN peptide (100 µg/ml), whereas intact laminin-1 had no effect. Importantly, uPA expression induced by elastase-generated laminin-1 fragments and the SN peptide were similar to that achieved with monocyte colony stimulating factor (75 ng/ml). Likewise, levels of MMP-9 activity in conditioned media derived from RAW264.7 macrophages incubated with elastase generated fragments or the SN peptide were similarly increased (Fig. 7). The observed increase in MMP-9 activity was further examined by determining the effect of elastase-generated fragments and SN peptide on steady state levels of MMP-9 mRNA. Following a 24-h incubation with laminin fragments or peptide, the levels of MMP-9 mRNA were markedly elevated over controls and cells incubated with intact laminin-1 (Fig. 7). Thus, the stimulation of MMP-9 activity was mirrored by an increase in MMP-9 gene activity.


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  		Fig. 6.   SN peptide up-regulates macrophage uPA expression. RAW264.7 cells were suspended in RPMI containing 10% FBS and aliquoted into 96-well plates (105/well). Following 4-6-h adherence, cells were washed to remove serum, and media were replaced with MSFM alone or MSFM containing intact laminin (50 µg/ml), elastase-generated laminin fragments (50 µg/ml), SN peptide (100 µg/ml), or MCSF (75 ng/ml). The next day, media were recovered and assayed for uPA activities as described under "Materials and Methods." The uPA data represent the means ± S.E. of three individual wells.


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  		Fig. 7.   SN peptide up-regulates macrophage MMP-9 activity and mRNA levels. RAW264.7 cells were suspended in RPMI containing 10% FBS and aliquoted into T25 flasks (5 × 106/flask). Following 4-6-h adherence, cells were washed to remove serum, and media were replaced with MSFM alone or MSFM containing intact laminin (50 µg/ml), elastase-generated laminin fragments (50 µg/ml) or SN peptide (100 µg/ml). The next day, conditioned media were recovered and assayed for MMP-9 activity by zymography. The poly(A) mRNA fractions were isolated from macrophage monolayers, and MMP-9 mRNA levels were determined by Northern blot hybridization utilizing a murine cDNA for MMP-9 as described under "Materials and Methods." For purposes of comparison, mRNA levels for constitutively expressed glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are presented.

We previously reported that the induction of macrophages proteinase expression by alpha 1:Ser2091-Arg2108 was dependent on a signaling pathway that resulted in the activation of MAPKerk1/2 (9). Treatment of cells with the MEK-1 inhibitor U0126 blocked the ability of alpha 1:Ser2091-Arg2108 to stimulate the phosphorylation/activation of MAPKerk1/2 and up-regulation of macrophage proteinase expression (9). Therefore, we determined whether the induction of proteinase expression by SN peptide was dependent on the activation of MAPKerk1/2. Serum-starved cells were incubated with SN peptide (100 µg/ml) for 0-20 min, and levels of phosphorylated MAPKerk1/2 were determined by Western blot. Incubation of cells with SN peptide resulted in a clear increase in levels of phosphorylated MAPKerk1/2 (Fig. 8A). Preincubation of macrophages with the MEK-1 inhibitor U0126 blocked SN peptide-induced uPA (Fig. 8B) and MMP-9 expression (Fig. 8C). Thus, these data demonstrate that two alpha 1-chain peptides from the tail region of laminin-1 trigger MAPK-dependent up-regulation of macrophage proteinase expression.


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  		Fig. 8.   Inhibition of MEK-1 blocks SN peptide induced uPA and MMP-9 expression. A, RAW264.7 macrophages (2 × 106/well) were cultured in RPMI medium without serum for 24 h. Cells were incubated with SN peptide (100 µg/ml) for 0-20 min, following which cell lysates were prepared. Phosphospecific Erk-1 (p44) and Erk-2 (p42) were identified by Western blot utilizing polyclonal anti-phosphospecific p44/42 MAPK as described under "Materials and Methods." B and C, macrophages (0.5 × 106/well) were preincubated for 30 min with 10 µM MEK-1 inhibitor U0126, following which 100 µg/ml SN peptide was added. Conditioned media (24 h) were collected and assayed for uPA and MMP activities as described under "Materials and Methods." The uPA data represent the means ± S.E. of three individual wells.

The stimulatory effect of elastase-generated laminin fragments and SN peptide on proteinase expression was confirmed utilizing thioglycollate elicited peritoneal macrophages (Fig. 9). When examined by zymography, the conditioned media derived from inflammatory macrophages contained both MMP-9 and MMP-2. As reported for RAW264.7 macrophages, MMP-9 activity was increased in media from macrophages incubated with elastase-generated laminin fragments or SN peptide. In contrast to MMP-9, the expression of MMP-2 was unaffected. uPA expression by thioglycollate-elicited peritoneal macrophages was markedly elevated (2), and the exposure of these cells to laminin fragments or alpha 1-chain peptides did not further stimulate their expression of uPA (data not shown). Thus, our observation that elastase-generated laminin fragments and the SN peptide stimulate proteinase expression by RAW264.7 macrophages is confirmed in primary macrophages.


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  		Fig. 9.   SN peptide up-regulates MMP-9 expression by peritoneal macrophages. Thioglycollate-elicited macrophages were cultured in RPMI-10% fetal calf serum overnight. Cells were washed to remove serum, and media were replaced with MSFM alone or MSFM containing intact laminin-1 (50 µg/ml), elastase-generated laminin fragments (50 µg/ml), or SN peptide (100 µg/ml). Following 24-h incubation at 37 °C, MMP activity in conditioned media was assessed by zymography as described under "Materials and Methods."

Laminin-1 Fragments Recovered from Abdominal Aortic Aneurysm Stimulate Proteinase Expression by Macrophages-- The regulatory role of laminin-1 on macrophage proteinase expression in vivo is unclear, since laminin-1 is rare in normal adult tissues (35). However, the expression of laminin-1 may increase under pathological conditions (36-41). Therefore, we determined whether laminin-1 fragments were present in an extract prepared from an abdominal aortic aneurysm and whether these fragments trigger macrophage proteinase expression. Aneurysms are inflammatory lesions characterized by elevated MMP and serine proteinase expression, which result in the degradation of vascular ECM and loss of structural integrity (10, 42). For this purpose, a tissue extract was prepared and fractionated on a column of heparin-Sepharose, as described for elastase-generated fragments of laminin-1. Unbound proteins (peak 1) were washed from the column and bound proteins (peak 2) were eluted with 0.5 M NaCl (Fig. 10A). The proteins in peaks 1 and 2 were analyzed by Western blot utilizing a polyclonal directed against a sequence in the tail of the alpha 1-chain that overlaps the peptide sequence previously reported to stimulate proteinase expression. As seen in Fig. 10B, anti-alpha 1:1856-2099 reacted strongly with the alpha 1-chain of intact laminin. The unfractionated extract, peak 1, and peak 2 contained proteins that were reactive with the anti-alpha 1-chain antibody. Peak 2 appears to contain intact and fragmented alpha 1-chain. The two prominent bands observed between 50 and 75 kDa in the tissue extract and peak 1 are nonspecific, since they appeared in blots probed with secondary antibody only (data not shown). Peaks 1 and 2 were dialyzed with DPBS and incubated with RAW264.7 macrophages overnight. uPA expression was stimulated severalfold by the peak 2 (heparin-binding) fraction, whereas incubation with the peak 1 fraction had no effect (Fig. 10C). Moreover, following immunodepletion with anti-alpha 1:1856-2099, peak 2 was unable to stimulate macrophages' uPA expression (Fig. 10D). In contrast, uPA expression by cells incubated with immunodepleted peak 1 was relatively unaffected. Likewise, immunoprecipitation with normal rabbit IgG had no effect on P2 induction of uPA expression (data not shown). Thus, these data support the hypothesis that fragments of alpha 1-chain, capable of stimulating macrophage proteinase expression, are generated in vivo. However, further studies are required to determine whether laminin fragments are present in normal abdominal aorta and if the observed fragmentation occurred ex vivo.


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  		Fig. 10.   Enhanced stimulation of macrophage uPA expression by heparin-binding laminin-1 fragments derived from aortic aneurysm. A, a tissue extract prepared from an aortic aneurysm was fractionated on a heparin-Sepharose column as described under "Materials and Methods." Unbound laminin fragments were washed from the column with wash buffer (peak 1). Bound fragments were eluted with wash buffer containing 0.5 M NaCl (peak 2). Fraction 5 of peak 1 and fractions 24 and 25 of peak 2 were collected. B, standard laminin-1 (Std Lmn), total extract (T), peak 1 (P1; fraction 5) and peak 2 (P2; fractions 24 and 25) were analyzed by Western blot utilizing polyclonal antibodies directed against alpha 1-chain amino acids 1856-2099 (anti-alpha 1:1856-2099) as described under "Materials and Methods." C, RAW264.7 cells were suspended in RPMI containing 10% FBS and aliquoted into 96-well plates (105/well). Following 4-6-h adherence, cells were washed to remove serum, and media were replaced with MSFM alone (Ctrl) or MSFM containing peak 1 (P1; 50 µg/ml), peak 2 (P2; 50 µg/ml), or MCSF (75 ng/ml). The next day, media were recovered and assayed for uPA activities as described under "Materials and Methods." The uPA data represent the means ± S.E. of three individual wells. D, peak 1 (P1) and peak 2 (P2) were incubated with anti-alpha 1:1856-2099 followed by Protein A-Sepharose as described under "Materials and Methods." Following centrifugation, the supernatants were recovered and tested for their ability to stimulate uPA expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ECM is a complex association of fibrillar proteins and adhesive glycoproteins, which provide structural stability to tissues and a substrate upon which cells adhere, move, and differentiate. The proteolysis of ECM, which occurs during development and under many pathologic conditions, can weaken the structural integrity of tissues, stimulate cellular invasion, trigger apoptosis or proliferation, and release matrix-bound growth factors (43). In addition, several lines of evidence suggest that ECM components contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules (17, 18, 43-49). Results of our previous studies, which determined whether the ECM regulates the macrophage-degradative phenotype, demonstrated that macrophages uPA and MMP-9 expression were stimulated in a dose-dependent manner by a synthetic peptide from the alpha 1-chain of laminin-1, whereas intact laminin-1 had no effect on proteinase expression (9). These data lead us to hypothesize that the domains of laminin-1, which stimulate macrophages' proteinase expression, are cryptic or assume a conformation that is not recognized by macrophages in the intact laminin molecule. Supporting this hypothesis was the observation that an extract of EHS-ECM, which stimulates macrophage proteinase expression, contains both intact laminin-1 and several laminin-1 fragments (9). When depleted of laminin and its fragments by immunoprecipitation with polyclonal anti-laminin-1 IgG, the EHS-ECM extract no longer stimulated macrophage proteinase activity (9). Results of experiments reported here demonstrate that selective cleavage of laminin-1 by elastase generates fragments that stimulate macrophage uPA and MMP-9 expression. These data are proof of the principle that proteolysis of laminin-1 generates fragments with new biologic activities.

Based on their affinity to heparin and reactivity with domain-specific antibodies, we conclude that the stimulatory domains exposed by elastase cleavage of laminin-1 are derived from the tail region of laminin-1. Utilizing synthetic peptides, we have identified two synthetic alpha 1-chain peptides from this region that stimulate macrophage expression of uPA and MMP-9: alpha 1:2091SRARKQAASIKVAVSADR2108 (or the hexapeptide alpha 1:2099SIKVAV2104) and alpha 1:2179SINNNRWHSIYITRFGNMGS2198. Both peptides are located in E8, an elastase-generated fragment of laminin-1 (14), which is the region of laminin primarily responsible for cell binding (50-52). The E8 fragment is derived from the long arm and consists of a coiled-coil (rodlike) region and G1-G3 of the COOH-terminal G-domain.

Despite the early observations that E8 fragments (50, 51) or anti-E8 (16) can block adhesion of cells to intact laminin-1, the peptide sequences in E8 responsible for cell binding remain controversial. In this regard, alpha 1:2091SRARKQAASIKVAVSADR2108 (or the hexapeptide alpha 1:2099SIKVAV2104) supports cell adhesion and stimulates a variety of biological responses including macrophage proteinase expression (8, 9, 53-56). Notwithstanding the number of diverse activities attributed to it, evidence suggests that alpha 1:2091SRARKQAASIKVAVSADR2108 is not exposed in intact laminin-1 (16). First, the alpha 1-chain peptide is derived from a portion of the alpha 1-chain associated with the coiled-coil portion of the E8 fragment. Second, anti-alpha 1:2099SIKVAV2104 has no effect on cell adhesion to the E8 fragment (19). Third, the binding of cells to intact laminin-1 and the E8 fragment was blocked by recombinant G-domain, which does not contain alpha 1:2091SRARKQAASIKVAVSADR2108 (16). Taken together, these data are consistent with our hypothesis that the domains in laminin that regulate macrophages' proteinase expression are cryptic in the intact molecule.

As discussed above, the binding of cells to laminin-1 appears to be mediated by the COOH-terminal portion of the alpha 1-chain, which extends past the coiled-coil region and forms the large oblong G-domain. It was previously demonstrated that alpha 1:2179SINNNRWHSIYITRFGNMGS2198 (SN peptide), located in the first loop of the G-domain, supports the adhesion of a variety of cells, and anti-SN peptide antibodies blocked the binding of cells to the E8 fragment (19). These data indicate that the SN peptide, unlike the proximal alpha 1:2091SRARKQAASIKVAVSADR2108, is exposed in intact laminin-1 and supports cell binding. However, in contrast to the SN peptide, intact laminin-1 fails to trigger MAPK activation and the up-regulation of uPA and MMP-9 expression. The divergent response of RAW264.7 and peritoneal macrophages to SN peptide versus intact laminin-1 indicates differential recognition mechanisms for these ligands. For example, intact laminin engages multiple receptors (12), which may act to suppress the signal triggered by the engagement of a single receptor by the SN peptide. Alternatively, the cell surface receptor(s) that recognize the SN peptide and initiate a signaling pathway that triggers proteinase may not recognize the SN peptide when its conformation is constrained as part of the intact molecule.

Results of experiments reported here provide clear evidence that selective proteolysis of laminin-1 generates fragments that up-regulate macrophages' proteinase expression. However, the regulatory role of laminin-1 fragments in macrophage-dependent tissue remodeling remains unclear, since laminin-1 expression is rare in normal adult tissues (35). In this regard, the expression of laminin-1 may increase under pathological conditions. For example, laminin-1 is one of several ECM proteins, which are elevated in following vascular injury and in atherosclerotic plaques (36-40). Likewise, laminin-1 is present in the adult kidney (57-59) and increases in immune complex glomerulonephritis (41). Supporting the hypothesis that stimulatory fragments of laminin-1 are present in pathologic specimens, we have demonstrated that an extract prepared from an abdominal aortic aneurysm contains immunoreactive alpha 1-chain fragments and up-regulates uPA expression by macrophages. The stimulatory activity in the tissue extract bound to heparin and was removed by immunoprecipitation with antibody directed against the alpha 1-chain.

In conclusion, the results of experiments reported here support the hypothesis that selective degradation of laminin-1 exposes cryptic domains that alter the macrophage-degradative phenotype through the up-regulation of uPA and MMP-9 expression. Based on heparin-Sepharose chromatography and mapping with antibodies, the stimulatory fragments are derived from the tail portion of laminin-1 involved in cell adhesion and binding to heparin sulfate proteoglycan. Two stimulatory alpha 1-chain peptides from this region have been identified that initiate a phosphorylation cascade resulting in the activation of MAPKerk1/2 and the up-regulation of proteinase expression. The exposure of cryptic domains in laminin-1 may play a role in regulation of macrophage proteinase expression and tissue remodeling at sites of injury and repair.

    ACKNOWLEDGEMENT

We acknowledge the technical assistance of Latanya Brandon.

    FOOTNOTES

* These studies were supported by National Institutes of Health Grants R01-HL40819 (to D. J. F.), R01-EY09747 (to G. W. L.), and R01-AG12712 (to T. A. M.) and Heritage Affiliate of the American Heart Association Grant-in-Aid 0150884T (to D. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Pathology, Rm. A678, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6491; Fax: 212-746-8789; E-mail: dfalcone@med.cornell.edu.

Published, JBC Papers in Press, February 4, 2002, DOI 10.1074/jbc.M111290200

    ABBREVIATIONS

The abbreviations used are: MMP, metalloproteinase; ECM, extracellular matrix; uPA, urokinase type plasminogen activator; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FBS, fetal bovine serum; RPMI, Roswell Park Memorial Medium; MSFM, macrophage serum-free medium; DPBS, Dulbecco's phosphate-buffered saline; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; EHS, Engelbreth Holm Swarm.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Werb, Z., Bainton, D. F., and Jones, P. A. (1980) J. Exp. Med. 152, 1537-1553[Abstract/Free Full Text]
2. Falcone, D. J., and Ferenc, M. J. (1988) J. Cell. Physiol. 135, 387-396[CrossRef][Medline] [Order article via Infotrieve]
3. Campbell, E. J., Cury, J. D., Shapiro, S. D., Goldberg, G. I., and Welgus, H. G. (1991) J. Immunol. 146, 1286-1293[Abstract]
4. Lassila, R. (1993) Eur. Heart J. 14, Suppl. k, 94-97
5. Shipley, J. M., Wesselschmidt, R. L., Kobayashi, D. K., Ley, T. J., and Shapiro, S. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3942-3946[Abstract/Free Full Text]
6. Carmeliet, P., Moons, L., Ploplis, V., Plow, E., and Collen, D. (1997) J. Clin. Invest. 99, 200-208[Medline] [Order article via Infotrieve]
7. Ploplis, V. A., French, E. L., Carmeliet, P., Collen, D., and Plow, E. F. (1998) Blood 91, 2005-2009[Abstract/Free Full Text]
8. Khan, K. M. F., and Falcone, D. J. (1997) J. Biol. Chem. 272, 8270-8275[Abstract/Free Full Text]
9. Khan, K. M. F., and Falcone, D. J. (2000) J. Biol. Chem. 275, 4492-4498[Abstract/Free Full Text]
10. Carmeliet, P., Moons, L., Lijnen, H. R., Baes, M., Lemaitre, V., Tipping, P., Drew, A., Eeckhout, Y., Shapiro, S., Lupu, F., and Collen, D. (1997) Nat. Genet. 17, 439-444[CrossRef][Medline] [Order article via Infotrieve]
11. Ramos-DeSimone, N., Hahn-Dantona, E., Sipley, J., Nagase, H., French, D. L., and Quigley, J. P. (1999) J. Biol. Chem. 274, 13066-13076[Abstract/Free Full Text]
12. Colognato, H., and Yurchenco, P. D. (2000) Dev. Dyn. 218, 213-234[CrossRef][Medline] [Order article via Infotrieve]
13. Kleinman, H. K., McGarvey, M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., and Martin, G. R. (1982) Biochemistry 21, 6188-6199[CrossRef][Medline] [Order article via Infotrieve]
14. Beck, K., Hunter, I., and Engel, J. (1990) FASEB J. 4, 148-160[Abstract]
15. Ott, U., Odermatt, E., Engel, J., Furthmayr, H., and Timpl, R. (1982) Eur. J. Biochem. 123, 63-72[Medline] [Order article via Infotrieve]
16. Yurchenco, P. D., Sung, U., Ward, M. D., Yamada, Y., and O'Rear, J. J. (1993) J. Biol. Chem. 268, 8356-8365[Abstract/Free Full Text]
17. Giannelli, G., Falk-Marzillier, J., Schiraldi, O., Stetler-Stevenson, W. G., and Quaranta, V. (1997) Science 277, 225-228[Abstract/Free Full Text]
18. Koshikawa, N., Giannelli, G., Cirulli, V., Miyazaki, K., and Quaranta, V. (2000) J. Cell Biol. 148, 615-624[Abstract/Free Full Text]
19. Chen, L. L., Shick, V., Matter, M. L., Laurie, S. M., Ogle, R. C., and Laurie, G. W. (1997) Am. J. Physiol. 272, L494-L503[Abstract/Free Full Text]
20. Raschke, W. C., Baird, S., Ralph, P., and Nakoinz, I. (1978) Cell 15, 261-267[CrossRef][Medline] [Order article via Infotrieve]
21. Edelson, P. J., and Cohn, Z. A. (1976) Purification and Cultivation of Monocytes and Macrophages in In Vitro Methods in Cell-mediated Tumor Immunity (Bloom, B. R. , and David, J. R., eds) , pp. 333-340, Academic Press, Inc., New York
22. Falcone, D. J., McCaffrey, T. A., Haimovitz-Friedman, A., and Garcia, M. (1993) J. Cell. Physiol. 155, 595-605[CrossRef][Medline] [Order article via Infotrieve]
23. Falcone, D. J., McCaffrey, T. A., and Vergilio, J.-A. (1991) J. Biol. Chem. 266, 22726-22732[Abstract/Free Full Text]
24. Masure, S., Nys, G., Fiten, P., Van Damme, J., and Ghislain, G. (1993) Eur. J. Biochem. 218, 129-141[Medline] [Order article via Infotrieve]
25. Schittny, J. C., and Yurchenco, P. D. (1990) J. Cell Biol. 110, 825-832[Abstract/Free Full Text]
26. Okada, Y., Morodomi, T., Enghild, J. J., Suzuki, K., Yasui, A., Nakanishi, I., Salvesen, G., and Nagase, H. (1990) Eur. J. Biochem. 194, 721-730[Medline] [Order article via Infotrieve]
27. Wilson, C. L., and Matrisian, L. M. (1996) Int. J. Biochem. Cell Biol. 28, 123-136[CrossRef][Medline] [Order article via Infotrieve]
28. Liotta, L. A., Goldfarb, R. H., and Terranova, V. P. (1981) Thromb. Res. 21, 663-673[CrossRef][Medline] [Order article via Infotrieve]
29. Moser, T. L., Enghild, J. J., Pizzo, S. V., and Stack, M. S. (1993) J. Biol. Chem. 268, 18917-18923[Abstract/Free Full Text]
30. Sung, U., O'Rear, J. J., and Yurchenco, P. D. (1997) Eur. J. Biochem. 250, 138-143[Medline] [Order article via Infotrieve]
31. Skubitz, A. P. N., McCarthy, J. B., Charonis, A. S., and Furcht, L. T. (1988) J. Biol. Chem. 263, 4861-4868[Abstract/Free Full Text]
32. Matter, M. L., and Laurie, G. W. (1994) J. Cell Biol. 124, 1083-1090[Abstract/Free Full Text]
33. Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590[Abstract/Free Full Text]
34. Nakahara, H., Nomizu, M., Akiyama, S. K., Yamada, Y., Yeh, Y., and Chen, W.-T. (1996) J. Biol. Chem. 271, 27221-27224[Abstract/Free Full Text]
35. Virtanen, I., Gullberg, D., Rissanen, J., Kivilaakso, J., Kiviluoto, T., Laitinen, L. A., Lehto, V. P., and Ekblom, P. (2000) Exp. Cell Res. 257, 298-309[CrossRef][Medline] [Order article via Infotrieve]
36. Dalferes, E. R., Jr., Radhakrishnamurthy, B., and Berenson, G. S. (1985) Artery 13, 41-49[Medline] [Order article via Infotrieve]
37. Nag, S. (1996) J. Neuropathol. Exp. Neurol. 55, 381-388[Medline] [Order article via Infotrieve]
38. Andresen, J. L., Rasmussen, L. M., and Ledet, T. (1996) Diabetes 45, S91-S94
39. Haruno, A., Morisaki, N., Ishii, I., and Saito, Y. (1999) Scand. J. Clin. Lab. Invest. 59, 395-403[CrossRef][Medline] [Order article via Infotrieve]
40. Glukhova, M., Koteliansky, V., Fondacci, C., Marotte, F., and Rappaport, L. (1993) Dev. Biol. 157, 437-447[CrossRef][Medline] [Order article via Infotrieve]
41. Peutz-Kootstra, C. J., Hansen, K., De, Heer, E., Abrass, C. K., and Bruijn, J. A. (2000) J. Pathol. 192, 404-412[CrossRef][Medline] [Order article via Infotrieve]
42. Freestone, T., Turner, R. J., Coady, A., Higman, D. J., Greenhalgh, R. M., and Powell, J. T. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 1145-1151[Abstract/Free Full Text]
43. Shapiro, S.